Human parainfluenza virus type 3 (HPIV-3) is an important respiratory tract pathogen capable of causing diseases such as pneumonia, bronchiolitis and croup in infants, young children, elderly and immunocompromised individuals. Currently, there are no effective antiviral therapies or vaccines against HPIV-3. The goal of this application is to gain a more complete understanding of the mechanisms through which HPIV-3 virus particles are assembled and released from cells. More specifically, the roles of multiple HPIV-3 proteins in promoting virus assembly and release will be determined (Specific Aim 1). By expressing HIPV-3 proteins individually and in various combinations in tissue culture cells and monitoring the release of virus-like particles (VLPs) containing these viral proteins, the contributions of various HPIV-3 proteins to virus assembly and release will be ascertained. Characterization of interactions between HPIV-3 proteins and host proteins during virus particle release will also be investigated (Specific Aim 2). One part of this Aim will be to identify the L domain on the HPIV-3 matrix (M) protein. The M proteins of HPIV-3 and related viruses trigger virus particle budding by binding and hijacking cellular proteins involved in vesicle formation. When this cellular machinery is brought to the cell membrane, virus particle budding/release from the cell occurs. M proteins bind these cellular proteins via short amino acid segments called late (L) domains. The HPIV-3 M protein does not have a known L domain. Thus, one goal of Specific Aim 2 is to identify the HPIV-3 M protein L domain. Another goal is identification of the cellular protein bound by the L domain of the M protein. Also, the role of Vps4, a cellular protein critical to the vesicle formation process, will be assessed. For most viruses containing L domains this protein is critical, but there is contradictory data regarding Vps4 involvement in budding of some viruses. By elucidating the role of this cellular protein in HPIV-3 budding, it is hoped that this contradictory data can be resolved. It should also be noted that VLP production could be an avenue for vaccine development, and identification of cellular proteins needed for virus replication could provide targets for drug development. PUBLIC HEALTH RELEVANCE: Human parainfluenza virus type 3 (HPIV-3) is a member of a group of viruses that causes significant worldwide morbidity and mortality. HPIV-3 itself is an important respiratory tract pathogen capable of causing diseases such as pneumonia, bronchiolitis and croup in infants, young children, elderly and immunocompromised individuals. Investigation into how HPIV-3 assembles new virus particles can provide leads toward the development of a vaccine or antiviral therapy against this virus.